Bacterial toxin ADP-ribosyltransferases (e.g., cholera toxin, pertussis toxin) appear to be responsible for the clinical presentation of some diseases due to bacterial infection. These toxin ADP-ribosyltransferases catalyze the transfer of the ADP-ribose moiety of NAD to critical regulatory proteins in mammalian cells. In the case of pertussis and cholera toxins, the ADP-ribose acceptors are the guanine nucleotide- regulatory (G) proteins shown to be involved in coupling of surface receptors to their intracellular effectors. The sequences of these ADP- ribosyltransferase and of a skeletal and cardiac muscle ADP- ribosyltransferases cloned earlier in the laboratory contain regions of amino acid identity and similarity, which, based on the crystallographic structure of the toxins, appear to be involved in the formation of the active site. RT6.2, a glycoprotein expressed specifically in postthymic lymphocytes, is glycophosphatidylinositol (GPI)-anchored; it has been implicated in some models of diabetes, although its role in the disease is unclear. In part, investigations into the function of RT6.2 have been hindered by the fact that the biochemical activity of RT6.2 was unknown. We observed that RT6.2, although at about 22-24 kDa, much smaller than the toxin and skeletal muscle ADP-ribosyltransferases, had significant regions of amino acid similarities, in particular, in those areas believed to be critical for enzymatic activity. To determine if RT6.2 catalyzed ADP-ribose transfer reactions, the gene was expressed in rat mammary adenocarcinoma cells and the activity of the expressed protein was examined. Cells transformed with RT6.2 expressed NAD glycohydrolase activity that was released from intact cells by phosphatidylinositol- specific phospholipase C, consistent with its presence on the cell surface. The molecular weight of the released enzyme was in good agreement with that of native RT6.2. These data suggest that the rat T cell alloantigen RT6.2 is a GPI-anchored NAD glycohydrolase.